The sex determining region Y (SRY) gene initiates sex differentiation
in man (1). A small number of cases of XY sex reversal have been described
which were due to mutations within the SRY gene, especially in the high
mobility group conserved region (HMG box) (2 and 3). We studied the DNA
sequence of the SRY gene in 22 Japanese in XY females (manuscript in preparation).
Here we report a case who has a single base pair substitution within the
HMG box.

The subject was a 28-year-old married Japanese woman with a history of
primary amenorrhea and infertility. Physical examination revealed an apparently
normal female with a weight of 62 kg and a height of 170.5 cm. While the
external genitals were those of a female, they were infantile with no hypertrophy
of the clitoris. The vagina was normal and a cervix was present. The uterus
was normal in shape and position. Laboratory examination revealed that the
amenorrhea was due to ovarian failure. There was no elevation of androgen
levels. Repeated chromosome analysis from leukocyte cultures consistently
showed a 46,XY karyotype. Both gonads were partially resected since the
risk of malignant development in XY gonadal dysgenesis has been reported
to be 25 % (4). The gonads consisted of fibroadipose tissue with no malignant
cells and nor with ovarian or testicular components. The diagnosis of XY
pure gonadal dysgenesis in this case is substantiated by the following features:
female phenotype, high stature (+2.9 S.D.), the presence of a uterus and
fallopian tubes and an apparently normal 46,XY karyotype.

Genomic DNA was prepared from the patient's leukocytes in peripheral
blood using the conventional method. The existence of the SRY gene was shown
by the polymerase chain reaction (PCR) as described elsewhere (5). We further
studied the gene with the single strand conformation polymorphism (SSCP)
method (6) and detected an abnormal band pattern (Fig. 1A).

Figure 1A. Detection of a mutation in the SRY gene. PCR-SSCP
analysis using a pair of primers spanning the entire HMG box. Each sequences
were: SRY 366: 5'-CAGTGTGAAACGGGAGAAAACAGT-3' SRY 367: 5'-CTTCCGACGAGGTCGATACTTATA-3'
One of the patients showed an abnormal band pattern (*) in electrophoresis
on a 6% acrylamide gel containing 10% glycerol.

Direct sequencing was carried out using, a biotinylated and a non-biotinylated
primers which enabled the use of a particular strand of a double-strand
DNA molecule for sequencing as only the biotinylated strands bind to the
streptavidin coated beads (7). Fig. 1B shows a single base substitution
in codon 107 counted from the initiation site of the SRY gene.

Figure 1. (B) PCR direct sequencing analysis. The left
is from a normal male control, and the right is from the patient. The arrow
indicates the nucleotide substitution G to A in the 107th codon from the
methionine initiation codon of the SRY gene.

Mutations in the SRY gene causing XY sex reversal have been reported
(2, 3, 8 and 9). Mutations causing a stop codon within the HMG box have
also been reported (10, 11 and 12). The mutation observed in this case also
caused a stop codon. However, it is in the middle of the HMG region and
different from other mutations reported in the literature (10, 11 and 12).

ACKNOWLEDGEMENTS

Supported by grants from the Faculty of Medicine, University of Tokyo,
the Ministry of Education, Science and Culture and the Ministry of Health
and Welfare, Japan.